RESUMEN
Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/toxicidad , Docilidad , Priones/química , Priones/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/toxicidad , Sitios de Unión de Anticuerpos , Calpaína/metabolismo , Cerebelo , Síndrome de Creutzfeldt-Jakob/metabolismo , Reactivos de Enlaces Cruzados , Mapeo Epitopo , Femenino , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/toxicidad , Técnicas In Vitro , Ligandos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Estrés Oxidativo , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPC/inmunología , Priones/genética , Especies Reactivas de Oxígeno/metabolismo , Eliminación de Secuencia/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/toxicidadRESUMEN
Misfolded prion proteins are the cause of neurodegenerative diseases that affect many mammalian species, including humans. Transmission of the prion diseases poses a considerable public-health risk as a specific prion disease such as bovine spongiform encephalopathy can be transferred to humans and other mammalian species upon contaminant exposure. The underlying mechanism of prion propagation and the species barriers that control cross species transmission has been investigated quite extensively. So far a number of prion strains have been characterized and those have been intimately linked to species-specific infectivity and other pathophysiological manifestations. These strains are encoded by a protein-only agent, and have a high degree of sequence identity across mammalian species. The molecular events that lead to strain differentiation remain elusive. In order to contribute to the understanding of strain differentiation, we have determined the crystal structures of the globular, folded domains of four prion proteins (cow, deer, elk and Syrian hamster) bound to the POM1 antibody fragment Fab. Although the overall structural folds of the mammalian prion proteins remains extremely similar, there are several local structural variations observed in the misfolding-initiator motifs. In additional molecular dynamics simulation studies on these several prion proteins reveal differences in the local fluctuations and imply that these differences have possible roles in the unfolding of the globular domains. These local variations in the structured domains perpetuate diverse patterns of prion misfolding and possibly facilitate the strain selection and adaptation.
Asunto(s)
Priones/química , Secuencia de Aminoácidos , Animales , Bovinos , Secuencia Conservada , Cricetinae , Cristalografía por Rayos X , Ciervos , Enlace de Hidrógeno , Proteínas Intrínsecamente Desordenadas/química , Mesocricetus , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.
Asunto(s)
Disacáridos/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Toxina Shiga II/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cristalografía por Rayos X , Disacáridos/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Toxina Shiga II/antagonistas & inhibidores , Toxina Shiga II/metabolismo , Análisis de Supervivencia , Toxemia/prevención & controlRESUMEN
Mucopolysaccharidosis type I (MPS I), caused by mutations in the gene encoding α-L-iduronidase (IDUA), is one of approximately 70 genetic disorders collectively known as the lysosomal storage diseases. To gain insight into the basis for MPS I, we crystallized human IDUA produced in an Arabidopsis thaliana cgl mutant. IDUA consists of a TIM barrel domain containing the catalytic site, a ß-sandwich domain and a fibronectin-like domain. Structures of IDUA bound to iduronate analogs illustrate the Michaelis complex and reveal a (2,5)B conformation in the glycosyl-enzyme intermediate, which suggest a retaining double displacement reaction involving the nucleophilic Glu299 and the general acid/base Glu182. Unexpectedly, the N-glycan attached to Asn372 interacts with iduronate analogs in the active site and is required for enzymatic activity. Finally, these IDUA structures and biochemical analysis of the disease-relevant P533R mutation have enabled us to correlate the effects of mutations in IDUA to clinical phenotypes.
Asunto(s)
Iduronidasa/química , Iduronidasa/metabolismo , Mucopolisacaridosis I/enzimología , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mucopolisacaridosis I/metabolismo , Conformación ProteicaRESUMEN
The serine/threonine PP-1c (protein phosphatase-1 catalytic subunit) is regulated by association with multiple regulatory subunits. Human ASPPs (apoptosis-stimulating proteins of p53) comprise three family members: ASPP1, ASPP2 and iASPP (inhibitory ASPP), which is uniquely overexpressed in many cancers. While ASPP2 and iASPP are known to bind PP-1c, we now identify novel and distinct molecular interactions that allow all three ASPPs to bind differentially to PP-1c isoforms and p53. iASPP lacks a PP-1c-binding RVXF motif; however, we show it interacts with PP-1c via a RARL sequence with a Kd value of 26 nM. Molecular modelling and mutagenesis of PP-1c-ASPP protein complexes identified two additional modes of interaction. First, two positively charged residues, Lys260 and Arg261 on PP-1c, interact with all ASPP family members. Secondly, the C-terminus of the PP-1c α, ß and γ isoforms contain a type-2 SH3 (Src homology 3) poly-proline motif (PxxPxR), which binds directly to the SH3 domains of ASPP1, ASPP2 and iASPP. In PP-1cγ this comprises residues 309-314 (PVTPPR). When the Px(T)PxR motif is deleted or mutated via insertion of a phosphorylation site mimic (T311D), PP-1c fails to bind to all three ASPP proteins. Overall, we provide the first direct evidence for PP-1c binding via its C-terminus to an SH3 protein domain.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida , Neoplasias/genética , Neoplasias/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteína Fosfatasa 1/genética , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Dominios Homologos srcRESUMEN
Sulfide:quinone oxidoreductase (SQR) is a peripheral membrane protein that catalyzes the oxidation of sulfide species to elemental sulfur. The enzymatic reaction proceeds in two steps. The electrons from sulfides are transferred first to the enzyme cofactor, FAD, which, in turn, passes them onto the quinone pool in the membrane. Several wild-type SQR structures have been reported recently. However, the enzymatic mechanism of SQR has not been fully delineated. In order to understand the role of the catalytically essential residues in the enzymatic mechanism of SQR we produced a number of variants of the conserved residues in the catalytic site including the cysteine triad of SQR from the acidophilic, chemolithotrophic bacterium Acidithiobacillus ferrooxidans. These were structurally characterized and their activities for each reaction step were determined. In addition, the crystal structures of the wild-type SQR with sodium selenide and gold(I) cyanide have been determined. Previously we proposed a mechanism for the reduction of sulfides to elemental sulfur involving nucleophilic attack of Cys356 on C(4A) atom of FAD. Here we also consider an alternative anionic radical mechanism by direct electron transfer from Cys356 to the isoalloxazine ring of FAD.
Asunto(s)
Acidithiobacillus/metabolismo , Quinona Reductasas/química , Quinona Reductasas/metabolismo , Cristalografía por Rayos X , Flavina-Adenina Dinucleótido/metabolismo , Sulfuro de Hidrógeno/metabolismo , Oxidación-Reducción , Relación Estructura-ActividadRESUMEN
In the presence of oligomeric chondroitin 4-sulfate (C4-S), cathepsin K (catK) forms a specific complex that was shown to be the source of the major collagenolytic activity in bone osteoclasts. C4-S forms multiple contacts with amino acid residues on the backside of the catK molecule that help to facilitate complex formation. As cathepsin L does not exhibit a significant collagenase activity in the presence or in the absence of C4-S, we substituted the C4-S interacting residues in catK with those of cathepsin L. Variants revealed altered collagenolytic activities with the largest inhibitory effect shown by the hexavariant M5. None of the variants showed a reduction in their gelatinolytic and peptidolytic activities when compared with wild-type catK, indicating no structural alteration within their active sites. However, the crystal structure of the M5 variant in the presence of oligomeric C4-S revealed a different binding of chondroitin 4-sulfate. C4-S is not continuously ordered as it is in the wild-type catK·C4-S complex. The orientation and the direction of the hexasaccharide on the catK surface have changed, so that the hexasaccharide is positioned between two symmetry-related molecules. Only one M5 variant molecule of the dimer that is present in the asymmetric unit interacts with C4-S. These substitutions have changed the mode of catK binding to C4-S and, as a result, have likely affected the collagenolytic potential of the variant. The data presented here support our hypothesis that distinct catK/C4-S interactions are necessary for the collagenolytic activity of the enzyme.
Asunto(s)
Catepsina K/química , Sulfatos de Condroitina/química , Colagenasas/química , Osteoclastos/enzimología , Multimerización de Proteína/fisiología , Sustitución de Aminoácidos , Catepsina K/genética , Catepsina K/metabolismo , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/metabolismo , Colagenasas/genética , Colagenasas/metabolismo , Cristalografía por Rayos X , Humanos , Mutación Missense , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The enzymes involved in the lysine biosynthetic pathway have long been considered to be attractive targets for novel antibiotics due to the absence of this pathway in humans. Recently, a novel pyridoxal 5'-phosphate (PLP) dependent enzyme called LL-diaminopimelate aminotransferase (LL-DAP-AT) was identified in the lysine biosynthetic pathway of plants and Chlamydiae. Understanding its function and substrate recognition mechanism would be an important initial step toward designing novel antibiotics targeting LL-DAP-AT. The crystal structures of LL-DAP-AT from Arabidopsis thaliana in complex with various substrates and analogues have been solved recently. These structures revealed how L-glutamate and LL-DAP are recognized by LL-DAP-AT without significant conformational changes in the enzyme's backbone structure. This review article summarizes the recent developments in the structural characterization and the inhibitor design of LL-DAP-AT from A. thaliana. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.
Asunto(s)
Proteínas Bacterianas/metabolismo , Transaminasas/metabolismo , Proteínas Bacterianas/química , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Especificidad por Sustrato , Transaminasas/químicaRESUMEN
Prion diseases are neurodegenerative diseases characterized by the conversion of the cellular prion protein PrP(c) into a pathogenic isoform PrP(sc). Passive immunization with antiprion monoclonal antibodies can arrest the progression of prion diseases. Here, the crystal structure of the Fab fragment of an antiprion monoclonal antibody, POM1, in complex with human prion protein (huPrP(c)) has been determined to 2.4 Å resolution. The prion epitope of POM1 is in close proximity to the epitope recognized by the purportedly therapeutic antibody fragment ICSM18 Fab in complex with huPrP(c). POM1 Fab forms a 1:1 complex with huPrP(c) and the measured K(d) of 4.5 × 10(-7) M reveals moderately strong binding between them. Structural comparisons have been made among three prion-antibody complexes: POM1 Fab-huPrP(c), ICSM18 Fab-huPrP(c) and VRQ14 Fab-ovPrP(c). The prion epitopes recognized by ICSM18 Fab and VRQ14 Fab are adjacent to a prion glycosylation site, indicating possible steric hindrance and/or an altered binding mode to the glycosylated prion protein in vivo. However, both of the glycosylation sites on huPrP(c) are positioned away from the POM1 Fab binding epitope; thus, the binding mode observed in this crystal structure and the binding affinity measured for this antibody are most likely to be the same as those for the native prion protein in vivo.
Asunto(s)
Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/química , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Proteínas PrPC/química , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Sitios de Unión de Anticuerpos , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Proteínas PrPC/inmunología , Enfermedades por Prión/inmunología , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
Acinetobacter baumannii is a common multidrug-resistant clinical pathogen that is often found in hospitals. The A. baumannii phosphoglycerate kinase (AbPGK) is involved in the key energy-producing pathway of glycolysis and presents a potential target for antibiotic development. AbPGK has been expressed and purified; it was crystallized using lithium sulfate as the precipitant. The AbPGK crystals belonged to space group P222(1). They diffracted to a resolution of 2.5â Å using synchrotron radiation at the Canadian Light Source.
Asunto(s)
Acinetobacter baumannii/enzimología , Fosfoglicerato Quinasa/química , Cristalización , Cristalografía por Rayos X , Expresión Génica , Fosfoglicerato Quinasa/genética , Fosfoglicerato Quinasa/aislamiento & purificaciónRESUMEN
AIMS: This aimed to study the causative agent, epidemiology, clinical characteristics, and treatment strategy targeting the main protease in porcine epidemic diarrhea. BACKGROUND: Porcine epidemic diarrhea (PED) is a contagious intestinal viral infection causing severe diarrhea, vomiting, and dehydration in pigs. High rates of mortalities and severe morbidities, approaching 100%, are reported in piglets infected with PEDV. In recent years, PED has been observed to influence the swine-farming nations in Europe, Asia, the USA, South Korea, and Canada. The PED virus (PEDV) transmission takes place through a faecal-oral route. OBJECTIVE: The objective is to review the characteristics of PEDV and its role in the disease. In addition, we aim to outline some possible methods to combat PED infection, including targeting the main protease of coronavirus and their future perspectives. METHODS: This study is a review of literature on the PED virus. RESULTS: Apart from symptomatic treatment and supportive care, there is no available specific treatment for PEDV. Appropriate disinfectants and cleaning are pivotal for the control of PEDV. To date, apart from anti-PEDV inhibitors, there are no specific drugs available commercially to treat the disease. Therefore, 3C-like protease (3CLpro) in PEDV that has highly conserved structure and catalytic mechanism serves as an alluring drug as it plays a vital role during viral polyprotein processing at the time of infection. CONCLUSION: A well synchronized and collective effort of scientists, swine veterinarians, pork industry experts, and associated authorities is essential for the accomplishment of proper execution of these required measures.
Asunto(s)
Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Animales , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/veterinaria , Diarrea/tratamiento farmacológico , Diarrea/epidemiología , Diarrea/veterinaria , Endopeptidasas , Péptido Hidrolasas , Porcinos , Enfermedades de los Porcinos/tratamiento farmacológico , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/prevención & controlRESUMEN
Scytalidoglutamic peptidase (SGP) is the prototype of fungal glutamic peptidases that are characteristically pepstatin insensitive. These enzymes have a unique catalytic dyad comprised of Gln(53) and Glu(136) that activate a bound water molecule for nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. The hydrolysis by SGP at peptide bonds with proline in the P(1)' position is a rare event among peptidases that we investigated using the series of fluorescence resonance energy transfer peptides, Abz-KLXPSKQ-EDDnp, compared with the series Abz-KLXSSKQ-EDDnp. The preference observed in these two series for Phe and His over Leu, Ile, Val, Arg, and Lys, seems to be related to the structure of the S(1) subsite of SGP. These results and the pH profiles of SGP activity showed that its S(1) subsite can accommodate the benzyl group of Phe at pH 4 as well as the positively charged imidazolium group of His. In the pH range 2 to 7, SGP maintains its structure and activity, but at pH 8 or higher it is irreversibly denatured. The intrinsic fluorescence of the Trp residues of SGP were sensitive to the titration of carboxyl groups having low pK values; this can be attributed to the buried Asp(57) and/or Asp(43) as described in SGP three-dimensional structure. The solvent kinetic isotope effects and the proton inventory experiments support a mechanism for the glutamic peptidase SGP that involves the nucleophilic attack of the general base (Glu(136)) activated water, and establish a fundamental role of the S(1) subsite interactions in promoting catalysis.
Asunto(s)
Hongos/enzimología , Glutamina/química , Péptido Hidrolasas/química , Serina Endopeptidasas/química , Ácido Aspártico/química , Carbono/química , Catálisis , Dicroismo Circular , Histidina/química , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Prolina/química , Conformación Proteica , Triptófano/químicaRESUMEN
Branched amino-acid biosynthesis is important to bacterial pathogens such as Mycobacterium tuberculosis (Mtb), a microorganism that presently causes more deaths in humans than any other prokaryotic pathogen (http://www.who.int/tb). In this study, the molecular cloning, expression, purification, crystallization and preliminary crystallographic analysis of recombinant IlvH, the small regulatory subunit of acetohydroxylic acid synthase (AHAS) in Mtb, are reported. AHAS carries out the first common reaction in the biosynthesis of valine, leucine and isoleucine. AHAS is an essential enzyme in Mtb and its inactivation leads to a lethal phenotype [Sassetti et al. (2001), Proc. Natl Acad. Sci. USA, 98, 12712-12717]. Thus, inhibitors of AHAS could potentially be developed into novel anti-Mtb therapies.
Asunto(s)
Acetolactato Sintasa/química , Mycobacterium tuberculosis/enzimología , Acetolactato Sintasa/genética , Acetolactato Sintasa/aislamiento & purificación , Secuencia de Aminoácidos , Cristalografía por Rayos X , Expresión Génica , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Mycobacterium tuberculosis (Mtb) acyl-CoA carboxylase is involved in the biosynthesis of mycolic acids, which are a key component of the bacillus cell wall. The Mtb genome encodes six acyl-CoA carboxylase ß subunits (ACCD1-6), three of which (ACCD4-6) are essential for survival of the pathogen on minimal medium. Mtb ACCD6 has been expressed, purified and crystallized. The two forms of Mtb ACCD6 crystals belonged to space groups P4(1)2(1)2 and P2(1)2(1)2(1) and diffracted to 2.9 and 2.5 Å resolution, respectively, at a synchrotron-radiation source.
Asunto(s)
Ligasas de Carbono-Carbono/química , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Ligasas de Carbono-Carbono/genética , Ligasas de Carbono-Carbono/aislamiento & purificación , Secuencia Conservada , Cristalografía por Rayos X , Expresión Génica , Datos de Secuencia Molecular , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Alineación de SecuenciaRESUMEN
Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP(c) to the pathogenic isoform PrP(sc). Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120-232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, ß = 95.6°.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/química , Priones/química , Animales , Cristalización , Cristalografía por Rayos X , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Priones/inmunologíaRESUMEN
The gene product of the open reading frame Rv3340 from Mycobacterium tuberculosis is annotated as encoding a probable O-acetylhomoserine (OAH) sulfhydrylase (MetC), an enzyme that catalyzes the last step in the biosynthesis of methionine, which is an essential amino acid in bacteria and plants. Following overexpression in Escherichia coli, the M. tuberculosis MetC enzyme was purified and crystallized using the hanging-drop vapor-diffusion method. Native diffraction data were collected from crystals belonging to space group P2(1) and were processed to a resolution of 2.1â Å.
Asunto(s)
Liasas de Carbono-Oxígeno/química , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/aislamiento & purificación , Secuencia Conservada , Cristalografía por Rayos X , Expresión Génica , Humanos , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Researchers from the world over are working to create prophylactic and therapeutic interventions to combat the COVID-19 global healthcare crisis. The current therapeutic options against the COVID-19 include repurposed drugs aimed at targets other than virus-specific proteins. Antibody-based therapeutics carry a lot of promise, and there are several of these candidates for COVID-19 treatment currently being investigated in the preclinical and clinical research stages around the world. The viral spike protein (S protein) appears to be the main target of antibody development candidates, with the majority being monoclonal antibodies. Several antibody candidates targeting the SARS-CoV-2 S protein include LY-CoV555, REGN-COV2, JS016, TY027, CT-P59, BRII-196, BRII-198 and SCTA01. These neutralizing antibodies will treat COVID-19 and possibly future coronavirus infections. Future studies should focus on effective immune-therapeutics and immunomodulators with the purpose of developing specific, affordable, and cost-effective prophylactic and treatment regimens to fight the COVID-19 globally.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , COVID-19/prevención & control , COVID-19/transmisión , Inmunoterapia/métodos , Pandemias/prevención & control , COVID-19/epidemiología , COVID-19/inmunología , HumanosRESUMEN
Streptogrisin B (SGPB) has served as one of the models for studying the catalytic activities of serine peptidases. Here we report its native crystal structures at pH 4.2 at a resolution of 1.18A, and at pH 7.3 at a resolution of 1.23A. Unexpectedly, outstanding electron density peaks occurred in the active site and the substrate-binding region of SGPB in the computed maps at both pHs. The densities at pH 4.2 were assigned as a tetrapeptide, Asp-Ala-Ile-Tyr, whereas those at pH 7.3 were assigned as a tyrosine molecule and a leucine molecule existing at equal occupancies in both of the SGPB molecules in the asymmetric unit. Refinement with relaxed geometric restraints resulted in molecular structures representing mixtures of the second tetrahedral intermediates and the enzyme-product complexes of SGPB existing in a pH-dependent equilibrium. Structural comparisons with the complexes of SGPB with turkey ovomucoid third domain (OMTKY3) and its variants have shown that, upon the formation of the tetrahedral intermediate, residues Glu192A to Gly193 of SGPB move towards the alpha-carboxylate O of residue P1 of the bound species, and adjustments in the side-chain conformational angles of His57 and Ser195 of SGPB favor the progression of the catalytic mechanism of SGPB.
Asunto(s)
Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Electrones , Genoma Bacteriano/genética , Enlace de Hidrógeno , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Serina Endopeptidasas/genética , Streptomyces griseus/enzimología , Streptomyces griseus/genética , Especificidad por SustratoRESUMEN
We have determined the X-ray crystal structure of the Mycobacterium tuberculosis (Mtb) gene product encoded by the open reading frame Rv0760c at 1.50 A resolution by single-wavelength anomalous dispersion (SAD) phasing of diffraction data from crystals of the selenomethionine-substituted protein. Refinement against diffraction data from the native protein resulted in R(work)=19.5% and R(free)=21.4%. The X-ray crystal structure shows that the homodimeric Rv0760c polypeptide has an alpha + beta conical barrel fold placing it among many structural neighbors of the nuclear transport factor 2 family (NTF2). This family is highly conserved in terms of structure; however the substrates and individual protein functions are diverse. The structures of native Rv0760c in several different crystal forms and Rv0760c bound to 17beta-estradiol 17-hemisuccinate (EH) have also been solved and analyzed.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Esteroide Isomerasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Estradiol/análogos & derivados , Estradiol/química , Estradiol/metabolismo , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Homología de Secuencia de Aminoácido , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismoRESUMEN
Haloalkane dehalogenases are enzymes well known to be important in bioremediation; the organisms from which they are produced are able to clean up toxic organohalides from polluted environments. However, besides being found in such contaminated environments, these enzymes have also been found in root or tissue-colonizing bacterial species. The haloalkane dehalogenase Rv2579 from Mycobacterium tuberculosis H37Rv has been cloned, expressed, purified and its crystal structure determined at high resolution (1.2A). In addition, the crystal structure of the enzyme has been determined in complex with the product from the reaction with 1,3-dibromopropane, i.e. 1,3-propanediol and in complex with the classical substrate of haloalkane dehalogenases, 1,2-dichloroethane. The enzyme is a two-domain protein having a catalytic domain of an alpha/beta hydrolase fold and a cap domain. The active site residues and the halide-stabilizing residues have been identified as Asp109, Glu133, His273, Asn39 and Trp110. Its overall structure is similar to those of other known haloalkane dehalogenases. Its mechanism of action involves an SN2 nucleophilic displacement.